An enduring principle for successful intervention in human disease is to identify - andthen prevent - the earliest steps in pathogenesis. In the case of Alzheimer's disease and its harbinger, mild cognitive impairment (MCI), studies from many labs support the still unproven hypothesis that the gradual accumulation and oligomerization of amyloid p-protein (A(3)in brain regions serving memory and cognition initiates this complex syndrome. Given the enormous resources being expended by academic and pharmaceutical scientists to identify anti-amyloid therapies and bring them to human trials, it is crucial to understand precisely how soluble A|3begins to oligomerize and whether this process actually induces the subtle compromise of synaptic function seen in MCI and early AD. In this new RO1 application, investigators who have collaborated productively to discover the natural secretion of low-n A|3oligomers in cell culture and then demonstrate their ability to inhibit long-term potentiation and disrupt memory in living animals now propose to rigorously define at the molecular level these earliest A(3assembly forms and elucidate their mechanisms of action on neuronal function. Based on extensive preliminary data and sensitive biochemical methods we have developed to isolate and study natural oligomers, we propose 4 interrelated Specific Aims. 1. Determine the precise molecular composition of naturally secretedA|3 oligomers by mass spectrometry and search for covalent crosslinks, associated small molecules and/or binding proteins that may contribute to their potent neuronal activity. 2. Characterize the effects of the natural oligomers on synaptic form and function, including in organotypic hippocampal cultures, and assess whether they can induce AD-type tau phosphorylation and altered transmitter release in vivo, 3. Purify the natural oligomers to homogeneity, intrinsically label them and identify their cognate molecular and cellular targets in living brain. 4. Assess 3 specific therapeutic strategies to decrease the production of cell-secreted oligomers and thereby abrogate their synaptotoxicity: (3- or y-secretase inhibitors; certain anti-aggregation compounds; and chaperone expression. Our extensive experience in studying this unlimited cellular source of physiological amounts of human A(3 oligomers should enable us to exploit this unique experimental paradigm to elucidate both the nature and the neuronal effects of the earliest A(3 assemblies, with attendant therapeutic implications. Relevance to Public Health: Because our central hypothesis is that the earliest-forming "oligomers" (doublets, triplets, etc.) of amyloid |3-protein underlie the subtle and progressive impairment of memory that is the hallmark of incipient AD, we will use a unique experimental system in which cultured cells naturally produce such early forms in order to decipher the precise nature of these pathogenic assemblies, identify their mechanism of injury on the neurons and synapses required for memory, and then block this process with novel drugs.